Molecular cloning, functional expression, and pharmacological characterization of 5-hydroxytryptamine3 receptor cDNA and its splice variants from guinea pig.
Polymerase chain reaction and rapid amplification of cDNA ends were used to isolate cDNAs encoding a 5-hydroxytryptamine3 (5-HT3) receptor subunit and its splice variants from guinea pig intestine. The amino acid sequence predicted from this cDNA is 81% homologous to the murine 5-HT3 receptor subunits cloned from NCB20 and N1E-115 cells. The splice variants code for two proteins differing by a deletion of six amino acids located in the large intracellular loop between transmembrane domains M3 and M4. For characterization, the cloned 5-HT3 cDNA was expressed in HEK 293 cells, and the electrophysiological and pharmacological properties of the recombinant ion/channel/receptor complex were investigated by patch clamping. Our data reveal that the cloned cDNAs code for guinea pig 5-HT3 receptors, which functionally assemble as homo-oligomers. The kinetic behavior of the ion channel and its sensitivity to several agonists and antagonists were markedly different from those of the cloned 5-HT3 receptors from mouse and human under similar experimental conditions. The agonists used were 5-hydroxytryptamine, 2-methyl-5-hydroxytryptamine, 1-phenylbiguanide (PBG), m-chlorophenylbiguanide, and the antagonists tropisetron and metoclopramide. In addition, 5-HT, PBG, and tropisetron were investigated through radioligand binding to isolated membranes. Compared with the human and murine 5-HT3 receptors, the guinea pig receptor showed prolonged desensitization kinetics. In addition, the guinea pig 5-HT3 receptor did not respond to the selective 5-HT3 receptor agonist PBG. Construction of chimeric receptors between guinea pig and human 5-HT3 receptor sequences localized the differences in desensitization kinetics to the carboxyl-terminal domain and the ligand binding site to the amino-terminal domain of the receptor protein. Molecular determinants of the PBG binding site of the human 5-HT3 receptor were localized to a 28-amino-acid spanning region adjacent to the M1 region.
Lankiewicz
S
S
Lehrstuhl für Zellphysiologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Lobitz
N
N
Wetzel
C H
CH
Rupprecht
R
R
Gisselmann
G
G
Hatt
H
H
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D006168:Guinea Pigs; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000235:genetics; D011985:Receptors, Serotonin / Q000187:drug effects / Q000235:genetics*; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014162:Transfection
D018076:DNA, Complementary; D007093:Imidazoles; D007211:Indoles; D012333:RNA, Messenger; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; C059068:GR 65630